Practical Skills in Biological Chemistry: Exhaustive Laboratory Guide

Electrophoresis and the Analysis of Blood Serum Proteins

Electrophoresis is defined as the capability of electrically charged proteins to move within an electric field. This laboratory methodology allows protein molecules to be separated based on the differences in their net charge. Specifically, molecules possessing a positive net charge migrate toward the cathode, while molecules with a negative net charge move toward the anode; those with no net charge do not undergo migration. For the electrophoretic division of blood serum, clinicians utilize special paper and a veronalic buffer maintained at a pH of $8.6$. At this specific alkaline pH, almost all blood serum proteins acquire a net negative charge and consequently migrate toward the anode. Following this separation and specific chemical treatment, a series of distinct fractions appear on the band including albumens and globulins.

The quantitative analysis of these protein fractions on an electrophoretical band is conducted through either photocolorimetric or densitometric methods. The resulting percentage ratio of blood serum proteins is referred to as a "proteinogramm." In a healthy individual, the normal proteinogramm values are as follows: albumens range from $53-62\%$, $\alpha_1$-globulins range from $2.7-5\%$, $\alpha_2$-globulins range from $7.4-10\%$, $\beta$-globulins range from $11-15\%$, and $\gamma$-globulins range from $15-21\%$. Deviations in these levels provide critical diagnostic data. Albumen levels increase during dehydration, shock, and hemoconcentration, while they decrease in cases of malnutrition, acute and chronic glomerulonephritis, acute and chronic hepatic failure, tumors, and leukemias.

Globulin levels exhibit specific clinical variations as well. An increase in globulins is associated with liver diseases such as viral hepatitis type A, liver cirrhosis, biliary cirrhosis, and hemochromatosis (hemosiderosis). Other conditions causing elevation include systemic lupus erythematosus, interstitial plasma cell myeloma, sarcoidosis, and various acute or chronic infectious diseases like typhus, leishmaniasis, malaria, and lymphogranuloma conditioned by venereal disease. Conversely, globulin levels decrease in states of poor nutrition, congenital agammaglobulinemia, acquired hypogammaglobulinemia, and lymphatic leukemia.

Quantification of Glucose and Transaminase Activity in Blood Serum

The normal concentration range for glucose in human blood serum is established between $3.33-5.55\,mmol/l$. The biochemical principle for determining glucose levels involves the enzyme glucose oxidase ($1.1.3.4.$), which catalyzes the oxidation of glucose into glucolactone, a reaction accompanied by the release of hydrogen peroxide ($H_2O_2$). The chemical reaction is represented as $Glucose + O_2 + H_2O \rightarrow Glucolactone + H_2O_2$. In the presence of peroxidase, the produced $H_2O_2$ oxidizes a chromogenic agent, such as 4-aminoantipyrine, to yield a colored pink product. The reaction follows: $H_2O_2 + 4\text{-aminoantipyrine} \rightarrow 2H_2O + \text{colored product (pink)}$. The optical density of this solution is then measured photometrically. Glucose levels rise (hyperglycemia) in diabetes mellitus, hyperthyrosis, adrenal cortex hyperfunction, hyperpituitarism (adenohypophysis hyperfunction), and occasionally liver diseases. Decreased levels (hypoglycemia) are seen in hyperinsulinism, adrenal cortex hypofunction, hypopituitarism, and during treatment with hypoglycemic medications.

Determining the activity of Aminotransferases, specifically Aspartate Aminotransferase (AsAT) and Alanine Aminotransferase (AlAT), is vital for diagnosing organ damage. Normal activity for AsAT is $0.028-0.139\,mcmol/l \times sec$ and for AlAT is $0.028-0.196\,mcmol/l \times sec$. These enzymes catalyze the transfer of an $\alpha\text{-amino}$ group from an $\alpha\text{-amino acid}$ (aspartate or alanine) to $\alpha\text{-ketoglutarate}$, forming glutamate alongside oxaloacetate or pyruvate. These products are converted into a yellow-colored product via reaction with $2,4\text{-dinitrophenylhydrazine}$. Increased activity of both enzymes occurs in liver cirrhosis, liver metastases, hepatitises (especially AlAT), and primary liver cancer. Myocardial infarction specifically elevates AsAT levels. AsAT also increases in muscular dystrophy and dermatomyositis. Activity decreases in vitamin $B_6$ deficiency, renal insufficiency, and during pregnancy. The De Rithis coefficient is noted as $1.33$.

Quantification of Urea, Creatinine, and Uric Acid

The normal concentration of urea in human blood serum is between $2.5-8.3\,mmol/l$. The determination principle relies on urease, which hydrolyzes urea into ammonia ($NH_3$) and carbon dioxide ($CO_2$). The ammonia is subsequently identified through a color reaction with sodium salicylate and alkaline hypochlorite. Blood urea levels increase due to renal failure (such as acute and chronic nephritis or urinary tract obstruction) and decreased renal blood flow (shock or adrenal cortex insufficiency). Levels decrease in cases of hepatic failure, nephrosis, and cachexia.

Creatinine quantification is performed using Yaffe’s test, with a normal range of $60-132\,mcmol/l$. In an alkaline medium, creatinine reacts with picric acid to form an orange-red coloration suitable for photometric measurement. Elevated creatinine levels are indicative of acute and chronic renal failure, urinary tract obstructions, or renal dysfunction induced by specific pharmaceuticals.

Uric acid levels are determined via the unitized phosphotungstic test, with normal serum values ranging from $0.12-0.4\,mmol/l$. The principle involves uric acid reducing the Foline reagent to produce a blue-colored product. Hyperuricemia (increased levels) is associated with gout, preeclampsia, eclampsia, leukemia, polycythemia, treatment with antileukemic drugs, renal failure, Lesch-Nyhan syndrome, and Down’s syndrome. Decreased levels are observed in acute hepatitis and during treatment with allopurinol.

Serum Calcium and Pyruvate Excretion Analysis

The normal concentration of calcium in human blood serum is $2.25-3.0\,mmol/l$. Its determination relies on Trilon B (EDTA), which chelates calcium ions, shifting the color of the indicator murexide from dark red to violet once all calcium is bound. Hypercalciemia can be physiological, as seen in newborns older than four days, or pathological, occurring in hyperthyroidism, vitamin D overdose, Itsenko-Cushing syndrome, acromegaly, leucosis, and cardiac decompensation. Hypocalciemia is observed in spasmophilia (infantile tetany), rickets, chronic nephritis, hypoparathyroidism, diarrhea, and acute pancreatitis.

Pyruvate, an intermediate of carbohydrate metabolism, is excreted in urine with a normal daily range of $1-25\,\mu g$ ($114-284\,\mu mol$). It reacts with $2,4\text{-dinitrophenylhydrazine}$ ($2,4\text{-DNPH}$) in a basic medium to form yellow-colored $2,4\text{-dinitrophenylhydrazone}$ of pyruvate, with color intensity proportional to concentration. Other hydrazones (from $\alpha\text{-ketoglutarate}$, oxaloacetate, or dihydroascorbic acid) are unstable in basic media and destroy rapidly. Pyruvate excretion increases in vitamin $B_1$ deficiency because thiamine pyrophosphate is a coenzyme of pyruvate dehydrogenase. Other causes for increase include diabetes mellitus, cardiovascular collapse, overproduction of adrenal medulla hormones, metabolic acidosis, and hypoxia. Pyruvate concentration decreases during narcosis.

Total Serum Cholesterol and Colloidal Stability Tests

Normal total serum cholesterol ranges from $4.5-6\,mmol/l$ (or $1.5-2\,g/l$). The measurement involves a sequence of enzymatic reactions: first, cholesterol esterase hydrolyzes cholesteryl esters into free cholesterol; second, cholesterol oxydase oxidizes cholesterol to cholestenon and releases $H_2O_2$; third, peroxidase catalyzes the oxidation of $H_2O_2$ to $O_2$ and $H_2O$. The emitted oxygen oxidizes colorless chromogens (4-aminoantipyrine and phenol) to a pink product. Cholesterol increases with aging (reflecting biological age), in atherosclerosis, cardiovascular diseases, gallstone disease, and hypothyroidism. Low cholesterol is found in cachexia, severe liver diseases, and hyperthyroidism.

Tests of colloidal stability measure the lability of serum proteins, which is determined by the ratio of coarse-dispersed to small-dispersed fractions (albumins to globulins). The normal ratio is $1.5-2.3$. Decreased stability is typically caused by an absolute or relative increase in $\gamma\text{-globulins}$. The Thymol test, based on the globulin-thymol-lipid complex formation in thymol-veronal buffer, has a normal range of $0-4\,\text{units}$. The Veltman's test involves the disturbance of stability in the presence of $CaCl_2$ under heating, with a norm of $1.3-1.5\,ml\,CaCl_2$.

Bilirubin, Katalase, Amylase, and Hemoglobin

Normal total serum bilirubin is $8.5-20\,mcmol/l$, with unconjugated bilirubin making up at least $75\%$ and conjugated bilirubin no more than $25\%$. In the Van den Bergh reaction, diazotized sulfonic acid reacts with bilirubin to form red azodipyrroles. Conjugated bilirubin (direct-reacting) reacts rapidly in aqueous solution, while unconjugated bilirubin (indirect-reacting) reacts slowly and requires a caffeine reagent to dissociate it from albumen. Hyperbilirubinemia is indicative of jaundices.

Katalase activity, an indicator of the antioxidant system, normally ranges from $11-20\,ED$. It is measured by titrating excessive $H_2O_2$ (not degraded by the enzyme) with potassium permanganate in an acidic medium: $5H_2O_2 + 2KMnO_4 + 3H_2SO_4 \rightarrow 8H_2O + 5O_2 + K_2SO_4 + 2MnSO_4$. Activity is expressed as milligrams of $H_2O_2$ decomposed by $1\,ml^3$ of blood over $30\text{ minutes}$.

Amylase activity in serum is normally $12-32\,mg/h \times ml$, and daily urine excretion is $44\,mg/l$. Amylase hydrolyzes polysaccharides like starch and glycogen. Elevated amylase in blood and urine is a primary indicator of pancreatitis (auto-digestion of the gland) or parothytis. Hemoglobin levels range from $120-140\,g/l$ in females and $130-160\,g/l$ in males. The principle involves red blood salt ($K_4[Fe(CN)_6]$) oxidizing $Fe^{2+}$ to $Fe^{3+}$ (methemoglobin), which reacts with acetoncyanhydrin to form brown hemoglobincyanade. Levels decrease in iron-deficient anemia and increase in dehydration and polycytemia.

Urine Analysis and Clinical Interpretation

Urinalysis evaluates several parameters. Normal color is pale to dark yellow; colorless urine may indicate long-term kidney disease or uncontrolled diabetes, mientras red urine suggests blood presence. Normal clarity is clear; cloudiness can be caused by white blood cells (pus), red blood cells, sperm, bacteria, yeast, mucus, or parasites such as trichomoniasis. Normal odor is slightly "nutty"; a sweet, fruity odor suggests uncontrolled diabetes, while a bad odor may indicate a UTI. A maple syrup odor points to maple syrup urine disease. Normal specific gravity is $1.005-1.030$. High specific gravity stems from dehydration or substances like sugar/protein, whereas low gravity suggests excessive fluid intake, severe kidney disease, or diuretic use.

Normal urine pH is $4.6-8.0$. High (alkaline) pH results from vomiting, kidney disease, UTIs, or asthma. Low (acidic) pH is caused by emphysema, uncontrolled diabetes, aspirin overdose, starvation, or ethylene glycol (antifreeze) consumption. Normal protein levels are negligible ($0.033 \text{ ‰}$); presence of protein indicates kidney damage, infection, cancer, lupus, heart failure, or preeclampsia. Glucose should normally be absent; its presence indicates diabetes, adrenal/liver/brain injury, or poisoning. Ketones should also be absent; their presence suggests uncontrolled diabetes, starvation, or isopropanol (rubbing alcohol) poisoning. Ketones are common after fasting for $18\text{ hours}$ or longer.

Summary of Manual Skills and Reference Values

Total serum protein ranges from $65-85\,g/l$. The lipid profile includes total lipids at $4-8\,g/l$, total cholesterol at $4-6.5\,mmol/l$ (recommended $\leq 5.2\,mmol/l$), HDL-cholesterol ($>1.2\,mmol/l$), and LDL-cholesterol ($<3.4\,mmol/l$). The Atherogenic coefficient calculation is: $\frac{\text{Total cholesterol} - \text{HDL cholesterol}}{\text{HDL cholesterol}}$, with a norm of $2-3\,U$. Triacylglycerols (TAGs) range from $0.55-1.65\,mmol/l$. Plasma lipoproteins include Chylomicrons ($0.1-0.4\,g/l$), VLDL ($0.8-1.5\,g/l$), LDL ($3-4.5\,g/l$), and HDL ($1.25-4.25\,g/l$).

Electrolyte and other serum values include: $Na^{+}$ ($130-157\,mmol/l$), and $K^{+}$ ($3.5-5.5\,mmol/l$). Immunoglobulins ranges are: $IgG$ ($8.1-18\,g/l$), $IgM$ ($0.6-2.8\,g/l$), and $IgA$ ($0.8-4.0\,g/l$). Glycated hemoglobin ($HbA1c$) should be $4-6\%$ of total $HbA$. The glucose threshold is $8-10\,mmol/l$. Additional urine values include urea ($20-35\,g/day$), and uric acid ($3.7-4.7\,mmol/day$). Clinical diagnostics focus on identifying diseases such as acute pancreatitis, myocardial infarction, and monitoring diabetes mellitus.